The present invention relates to a method for the determination of enzyme activity and a composition suitable therefor. In the present specification, the enzymes to be determined are leucine aminopeptidase (hereinafter referred to as LAP) and .gamma.-glutamyltranspeptidase (hereinafter referred to as .gamma.-GTP).
Heretofore, as a method for measuring the enzyme activities, it has been known that a substrate for enzymes obtained by binding leucine or glutamic acid with a chromogen is decomposed by the action of the enzyme in a sample to form a colored compound which is determined.
As the chromogen, p-nitroanilide, .beta.-naphthylamide, 4-hydroxy-3-carboxyanilide, p-hydroxyanilide, 4-N,N-dialkylamino-3-carboxyanilide, 4-N,N-dialkylaminoanilide and the like are used.
The solubility of these compounds is, at most, 6.5 mg/ml and the rate of enzymatic reaction is controlled by the rate of solubility. Further, in the determination of the pigment formed by using these substrates, the results are likely to be influenced by the other components in the sample. Because the determination is made by measuring the absorbancy in the ultraviolet region, a enzymatic reaction must be carried out under the strong alkaline condition which is a dangerous condition. The sensitivity is low because the molecular extinction coefficient of the formed pigments is low a long period of time is required to complete the enzymatic reaction, and thus the accuracy of the results by the method is low.
As a result of studies of substrates having few faults, it has been found that p-aminoanilide compounds wherein an alkyl or hydroxyalkyl group containing a sulfo group is bonded to the amino group in the para-position, have excellent solubility and stability of the pigment formed by the enzymatic reaction. It is possible to measure in the visible region because the chromogen has a high molecular extinction coefficient.